DESCRIPTION: Mechanical forces play increasingly recognized roles in directing T cell activation and subsequent function. We recently discovered that T cells are sensitive to the rigidity of an underlying substrate presenting activating ligands to CD3 and CD28. Specifically, primary mouse CD4+ T cells exhibited increased activation on surfaces presenting antibodies to CD3 and CD28 as material rigidity increased. While established in other cell systems, predominantly in the context of integrin-ECM and cadherin-cadherin interactions, mechanosensing by T cells through the CD3/TCR and CD28 is not well understood. Addressing this gap in knowledge would provide a new system upon which the mechanosensing concepts developed in other systems could be tested, as well as provide new insight into T cell physiology and advanced tools for immunotherapy. The proposed study seeks to address the issue that T cell-substrate interaction, like other cells, occurs in multiple stages. The ability t control the rigidity of the substrate during each stage would reveal the role each one has in mechanosensing, focusing subsequent studies into the molecular pathways responsible for this ability. Such knowledge could provide new targets for replicating or improving upon the benefits of soft substrates, but using chemical or biological agents. Towards this goal, we propose a magnetically actuated system that provides on-demand, reversible, and repeatable control over the mechanical stiffness presented to an adherent cell. We first focus on development and validation of this new system, which is an adaptation of the elastomer pillar array technology used for traction force microscopy. This system is then used to independently assess T cell mechanosensing during early cell spreading and sustained contraction. In a complementary direction, we will also assess dynamics of CD3/TCR signaling as a function of substrate rigidity, a core concept in a developing model of T cell mechanosensing. Successful completion of the proposed study will be a key advance in the emerging interdisciplinary field of T cell mechanobiology. In addition, it is expected that the proposed system will be immediately and directly applicable towards other cellular systems.